Hematopoietic stem cells (HSCs) are rare cells that have the unique ability to self-renew and differentiate into cells of all hematopoietic lineages. The expansion of HSCs has remained an important goal to develop advanced cell therapies for bone marrow transplantation and many blood disorders. During the last two to three decades, in which the first hematopoietic growth factors were identified, there have been numerous attempts to expand HSCs in vitro using purified growth factors that are known to regulate HSCs. However, these attempts have been met with limited success for clinical applications. An innovative approach is urgently needed for the research community to succeed in unraveling HSC expansion biology and creating a breakthrough in the ability to expand HSCs in vitro to clinically useful numbers. This would have tremendous impact in the areas of bone marrow transplantation and gene therapy for hematologic cancers and disorders. Toward this end, we have made several striking discoveries that shed new light on HSC expansion using cytokine-dependent SALL4 technology and lay the groundwork for the studies proposed here. We hope our studies will uncover an entirely new form of HSC expansion that could be developed and it is thus feasible to translate this study into the clinical setting. The Specific Aims of our proposal are focused on achieving this objective. Aim 1. To define the kinetics, duration, and magnitude of SALL4-induced enhancement of HSC expansion ex vivo and in vivo. Aim 2-To compare two different approaches to expand human umbilical cord (hUBC) stem cells and characterize the extent of their contribution of short-term and long-term repopulation to marrow recovery. Aim 3. - To identify proteins that specifically interact with the N-terminal domains of SALL4 by taking advantage of protein mass spectrometry from HSCs.